The mechanisms underlying cellular injury following hypoxia (ischemia) and reoxygenation (reflow) will be examined in the isolated, perfused, hemoglobin-free rat liver. Preliminary experiments have shown that within 15 minutes of the onset of hypoxia, structural alterations appear which are characterized by bleblike protrusions of hepatocyte plasma membrane and cytoplasm through fenestrations of the endothelium. Reoxygenation caused the release of these blebs and severe disruption of cellular volume regulation. The role of calcium, the cytoskeleton, and other factors in the onset of and recovery from hypoxic cell injury will be studied utilizing ultrastructural, biochemical and biophysical techniques. Cell structure, especially of the cell surface and the cytoskeleton, will be examined by light microscopy, immunocytochemistry, and thin section, scanning, freeze fracture and intermediate high voltage electron microscopy. Sublobular oxygen tensions will be measured using mini-oxygen electrodes. Sublobular fluorescence of NAD(P)H (an intrinsic probe of tissue oxygenation), chlorotetracycline (a calcium probe) and rhodamine 123 (a probe of mitochondrial membrane potential) will be measured using micro-light guides. Whole liver metabolism will be monitored by the inflow-outflow difference of oxygen, pH, lactate, pyruvate, and other metabolites. The mechanisms of hypoxic cellular injury will be elucidated by systematically altering the composition of the perfusate (e.g. varying pH, osmotic and oncotic pressure, calcium ion), by varying flow parameters, and by using pharmacological agents which have specific effects on calcium transport across the membrane, calmodulin activity, cytoskeletal structure, and cellular bioenergetics. These factors will be related to the mechanism(s) of enzyme release by injured liver. The proposed studies will provide essential new information concerning the basic mechanisms underlying cellular injury from ischemic disease.